At 17:50 07/07/00 +0200, Dominic-Luc Webb wrote:
>It appears the reason for the confusion about
>whether PFK is rate limiting depends on the
>cell type. The nay sayers are citing yeast
>and trypanosomes, while I am focused on mammalian
>PFK studies, largely in skeletal muscle and
>pancreatic beta cells. In the latter case, it
>appears to remain valid that PFK is effectively
>rate limiting.
The important point here is to establish what characteristics you believe
that a rate-limiting enzyme has, and to show that PFK exhibits those
properties. I think you will have a hard time of it, but I wait to see
what you offer. I should make it clear that evidence that PFK is a
regulated enzyme does not count, because that is equally true of yeast
PFK. Nor is it sufficient to show existence of a cross-over effect at PFK,
because that also is only related to regulatory properties and yeast PFK
shows a beautiful cross-over on the aerobic-anaerobic transition.
Some additional things you need to consider:
1. It's not just yeast and trypanosomes. 30 fold over-expression of PFK
in potato tubers has no effect on glycolytic rate. Furthermore, this
negligible effect on glycolysis is entirely consistent with Metabolic
Control Analysis theory, which does predict the effect that the
over-expression has on glycolytic metabolite concentrations. (Thomas et al,
1997,
Biochem. J., 322, 119--127).
2. Over-expression of PFK in Chinese Hamster Ovary cells (yes - a
mammalian system) has no significant effect on glycolytic flux according to
Ana Urbano (PhD thesis, Cambridge). The same work showed the reasons why
some earlier studies on mammalian cells had been mistaken on this issue.
3. The fact that an enzyme regulated by feedback effects would not be
rate-limiting was a theoretical prediction of Kacser & Burns (1973). The
experimental results for yeast, potato and CHO cells could have invalidated
this theory, but they didn't, they were compatible. The same theory forms
the grounds why many of us believe that PFK isn't rate-limiting in
mammalian cells either. Why do you think the theory got it right for some
cells, but won't for mammalian cells?
4. Without starting a mini-review, the evidence from theory and experiment
is that regulated enzymes (generally) are more effective at ensuring
metabolite homeostasis at low metabolite levels than at controlling
flux. The evidence relevant to PFK involves replacing it in yeast with a
non-allosteric version from Dictyostelium discoideum (Estevez et al, 1995,
FEBS Lett, 374, 100--104), and a version insensitive to F26BP (Boles, E. et
al, 1996, Mol. Microbiol., 20, 65--76), both without effect on glycolytic
rate, but the latter causing large changes in metabolite concentrations.
5. The expected result of activating a single enzyme in a pathway is that
the effect on the metabolic rate will be generally disappointing (Small and
Kacser,1993, Eur. J. Biochem., 213, 613--624), but it will have larger
effects on metabolite concentrations. This is contrary to the observed
effects in physiological conditions. In animal skeletal muscle in
particlular, increases in glycolytic rate of several hundred fold are
accompanied by negligible changes in glycolytic metabolites (examples are
given in Thomas and Fell, 1998, Advan. Enzyme Regul., 38, 65--85). We can
give metabolic control analysis arguments that this is inconsistent with
glycolytic control by PFK (Thomas & Fell, 1998, Eur. J. Biochem., 258,
956--967). However, you don't have to be a believer in Metabolic Control
Analysis to see that there's a problem (see Hochachka, 1994, "Muscles as
Molecular and Metabolic Machines" CRC Press, Boca Raton). Any explanation
of metabolite homeostasis in a metabolic pathway will have to involve
control mechanism acting along the length of the pathway.
Anyway, over to you, and anyone else out there who wants to join in: what
do you think are the characteristics of a rate-limting enzyme, and what is
the evidence that mammalian PFK has them whilst yeast PFK does not?
>It is sad that there were so many unprofessional
>responses in this group and that nobody suggested
>this might be the reason for these different
>views. Instead, some of you chose to slander
>very good biochemists, making this a poor excuse
>for a scientific exchange.
From the perspective of someone who has examined a lot of PhD candidates,
I think Benno ter Kuile's comments to you were kindly meant. When you have
to defend your thesis, you're likely to need to show you know the
arguments on both sides; believing a big name isn't enough. It's not
slander to say a scientist is wrong; progress in science consists of
replacing earlier views, and the fate of most of us will be to have our
ideas superceded by better ones. The best we can do is attempt to aid
progress, not hinder it.
David Fell
===================================================
Prof David A Fell,
School of Biological & Molecular Sciences, Oxford Brookes University,
Headington, Oxford OX3 0BP, UK
Tel: +44 (0)1865 483247 Fax: +44 (0)1865 484017
http://bms-mudshark.brookes.ac.uk/
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